Donatella De Feo

iPSC-derived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF

The possibility of generating neural stem/precursor cells (NPCs) from induced pluripotent stem cells (iPSCs) has opened a new avenue of research that might nurture bench-to-bedside translation of cell transplantation protocols in central nervous system myelin disorders. Here we show that mouse iPSC-derived NPCs (miPSC-NPCs)-when intrathecally transplanted after disease onset-ameliorate clinical and pathological features of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Transplanted miPSC-NPCs exert the neuroprotective effect not through cell replacement, but through the secretion of leukaemia inhibitory factor that promotes survival, differentiation and the remyelination capacity of both endogenous oligodendrocyte precursors and mature oligodendrocytes. The early preservation of tissue integrity limits blood-brain barrier damage and central nervous system infiltration of blood-borne encephalitogenic leukocytes, ultimately responsible for demyelination and axonal damage. While proposing a novel mechanism of action, our results further expand the therapeutic potential of NPCs derived from iPSCs in myelin disorders.

Commonalities in immune modulation between mesenchymal stem cells (MSCs) and neural stem/precursor cells (NPCs)

Owing to their unique immunomodulatory properties, mesenchymal stem cells (MSCs) have been advocated as a potential therapy for numerous pathological conditions in which immune-mediated inflammatory reactions play a crucial role, such as autoimmune disorders, cerebrovascular diseases and tumours. Increasing evidence suggest that stem cells, other than MSCs, are also capable of immunomodulation. Neural stem/precursor cells (NPCs) have been among the first stem cells to show immunomodulatory properties and nowadays represent one the most studied and promising stem cell subtype in still uncurable acute and chronic inflammatory neurological disorders. Although the ontogeny of NPCs and MSCs greatly diverges, their immunomodulatory mechanisms are similar and are largely based on the bystander (paracrine) effect through membrane-bound and soluble mediators that influence the behavior of host immune cells. This observation suggests the existence of a core stem cell signature across different stem cell lineages and that shared signalling pathways between the stem cell niche and the inflammatory immune response likely mediate both NPC and MSC immunomodulatory effect.

Delayed Diagnosis in Pediatric Headache: An Outpatient Italian Survey

(Headache 2011;51:1267‐1273)

Myeloid cells as target of fingolimod action in multiple sclerosis

Objective: To track the effects of fingolimod, an approved drug for multiple sclerosis (MS), on the activation of myeloid cells from the periphery to the CNS. Methods: In vitro and ex vivo immunologic studies coupled with flow cytometry were performed to evaluate the action of fingolimod on lipopolysaccharide (LPS)–induced expression of activation markers in human monocytes from healthy participants, participants with untreated MS, and participants with fingolimod-treated MS. In vivo administration of fingolimod during experimental autoimmune encephalomyelitis (EAE) was established to verify the activation state of splenic, CNS infiltrating, and CNS resident myeloid cells ex vivo at flow cytometer. Results: We found that in vitro exposure of human monocytes to fingolimod inhibited LPS-induced CD25 and CD150 expression and tumor necrosis factor–α (TNF-α) secretion without altering immune cell survival. Further, EAE treatment with fingolimod led to reduced amounts of TNF-α produced by myeloid cells in vivo in the spleen and CNS. Finally, while displaying normal induction of CD25 and CD150 levels at high LPS concentration, monocytes from patients with fingolimod-treated MS showed significantly higher activation threshold at suboptimal LPS stimulation than controls. Conclusions: The inhibition of myeloid cell activation may be part of the immunosuppressive action of fingolimod and take place in the periphery and in the CNS.

Neural precursor cell–secreted TGF-β2 redirects inflammatory monocyte-derived cells in CNS autoimmunity

In multiple sclerosis, the pathological interaction between autoreactive Th cells and mononuclear phagocytes in the CNS drives initiation and maintenance of chronic neuroinflammation. Here, we found that intrathecal transplantation of neural stem/precursor cells (NPCs) in mice with experimental autoimmune encephalomyelitis (EAE) impairs the accumulation of inflammatory monocyte-derived cells (MCs) in the CNS, leading to improved clinical outcome. Secretion of IL-23, IL-1, and TNF-&agr;, the cytokines required for terminal differentiation of Th cells, decreased in the CNS of NPC-treated mice, consequently inhibiting the induction of GM-CSF–producing pathogenic Th cells. In vivo and in vitro transcriptome analyses showed that NPC-secreted factors inhibit MC differentiation and activation, favoring the switch toward an antiinflammatory phenotype. Tgfb2–/– NPCs transplanted into EAE mice were ineffective in impairing MC accumulation within the CNS and failed to drive clinical improvement. Moreover, intrathecal delivery of TGF-&bgr;2 during the effector phase of EAE ameliorated disease severity. Taken together, these observations identify TGF-&bgr;2 as the crucial mediator of NPC immunomodulation. This study provides evidence that intrathecally transplanted NPCs interfere with the CNS-restricted inflammation of EAE by reprogramming infiltrating MCs into antiinflammatory myeloid cells via secretion of TGF-&bgr;2.

A strange case of waitress headache

In September, 2010, a 21-year-old woman, who worked as a waitress at an Italian restaurant, presented to our neuro-logical department with a sudden-onset, non-specifi c headache of moderate severity, which was unresponsive to over-the-counter medications. She also had vomiting and unsteadiness. 2 weeks before she had had a self-limited episode of febrile gastroenteritis. On admission, she was afebrile and she had horizontal nystagmus. Blood tests showed slight leuco cytosis. Over the next 24 h, her neurological status worsened and she developed mild neck stiff ness, bilateral cerebellar ataxia, and right-sided facial hypoaesthesia. After 48 h, she had dysphagia, dysarthria, and persistent hiccupping. MRI of the brain showed large brainstem and cerebellar lesions with patchy regions of gadolinium enhancement and relevant oedema (fi gure). Cerebrospinal fl uid (CSF) analysis showed: protein concen-tration 1·38 g/L, glucose concentration 2·26 mmol/L, leucocytes 10/μL (70% poly morphonuclear). Oligoclonal bands were absent and CSF Gram staining negative. Molecular and serological tests for herpes simplex virus, cytomegalovirus, Epstein-Barr virus, entero virus,

Selective killing of spinal cord neural stem cells impairs locomotor recovery in a mouse model of spinal cord injury

BackgroundSpinal cord injury (SCI) is a devastating condition mainly deriving from a traumatic damage of the spinal cord (SC). Immune cells and endogenous SC-neural stem cells (SC-NSCs) play a critical role in wound healing processes, although both are ineffective to completely restore tissue functioning. The role of SC-NSCs in SCI and, in particular, whether such cells can interplay with the immune response are poorly investigated issues, although mechanisms governing such interactions might open new avenues to develop novel therapeutic approaches.MethodsWe used two transgenic mouse lines to trace as well as to kill SC-NSCs in mice receiving SCI. We used Nestin CreERT2 mice to trace SC-NSCs descendants in the spinal cord of mice subjected to SCI. While mice carrying the suicide gene thymidine kinase (TK) along with the GFP reporter, under the control of the Nestin promoter regions (NestinTK mice) were used to label and selectively kill SC-NSCs.ResultsWe found that SC-NSCs are capable to self-activate after SCI. In addition, a significant worsening of clinical and pathological features of SCI was observed in the NestinTK mice, upon selective ablation of SC-NSCs before the injury induction. Finally, mice lacking in SC-NSCs and receiving SCI displayed reduced levels of different neurotrophic factors in the SC and significantly higher number of M1-like myeloid cells.ConclusionOur data show that SC-NSCs undergo cell proliferation in response to traumatic spinal cord injury. Mice lacking SC-NSCs display overt microglia activation and exaggerate expression of pro-inflammatory cytokines. The absence of SC-NSCs impaired functional recovery as well as neuronal and oligodendrocyte cell survival. Collectively our data indicate that SC-NSCs can interact with microglia/macrophages modulating their activation/responses and that such interaction is importantly involved in mechanisms leading tissue recovery.

Assessing the role of innovative therapeutic paradigm on multiple sclerosis treatment response

Within the last decade, many changes have been made to the management of patients with multiple sclerosis (MS). The aim of our study was to investigate the global impact of all these changes on the diseases course.

Influence of experimental autoimmune encephalomyelitis on the localization and viability of neural stem cells after intrathecal transplantation

Introduction: Murine bone marrow mesenchymal stem cells (MSC) can adapt their therapeutic effects to specific microenvironments. In inflammatory conditions, in vitro MSC showed a modulation of genes referable to interferon gamma (IFNg) stimulation, likely involved in the enhancement of the therapeutic plasticity of MSC. The identity of proteins mediating this plasticity is not fully elucidated yet. This study aims at identifying the changes in the protein profile in MSC under IFNg stimulation that can explain the increased neuroprotective and immunomodulatory properties of MSC in inflammatory conditions. Materials and Methods: A Stable Isotope Labeling with Amino acids in cell Culture (SILAC)-based proteomic approach was used. The cells were labeled through the metabolic incorporation of isotopic 13C6Lysine and 13C6-Arginine or 12C6-Lysine and 12C6-Arginine into the proteins expressed by IFNg-primed or unprimed MSC, respectively. Protein identification and quantitation were performed by LC–ESIMS/MS with the Velos Elite Orbitrap mass spectrometer coupled to Mascot Distiller 2.3 interface. The changes in protein expression and signaling pathways represented in the two conditions were analyzed with the Ingenuity Pathways Analysis software. Results: Analysis of the IFNg-primed MSC lysates highlighted an activation of the antigen presentation process, lipid metabolism and response to oxidative stress. Protein synthesis, cell cycle regulation, endoplasmic reticulum stress, along with IFNg-signaling, were also perturbed. IFNg-primed MSC conditioned medium showed an increased level of proteins involved in the regulation of cell–cell and cell–matrix interactions, immunomodulatory and neuroprotective proteins and proteins involved in cholesterol trafficking (analysis in progress). Furthermore, preliminary data suggest that extracellular membrane vesicles released byMSC could shuttle relevantmolecules to mediate, at least partially, the immunomodulatory effect of MSC on activated T cells. Discussion and Conclusions: In response to inflammatory stimulation, MSC are able to change profoundly the protein profile and activate specific metabolic and signaling pathways that likely lead to the enhancement of their therapeutic effects. The identification of new regulatory molecules involved in the therapeutic plasticity of MSC paves the way to the study of new therapeutic approaches to treat autoimmune and neurodegenerative diseases.

Subarachnoid neurocysticercosis with spinal involvement presented with headache

A 49-year-old man was referred to our clinic for a 1-year history of severe lumbar pain irradiating to the lower limbs and left foot drop causing progressive gait impairment. Six years before, he had had for several months weekly nocturnal attacks of bilateral throbbing headache associated with neck stiffness lasting 1–2 h that had disappeared in the following 3 years. This late-onset headache, associated to danger signals such as neck stiffness and nocturnal exacerbations, was suggestive of a secondary headache. Indeed, on the brain CT performed at that time two small parenchymal calcifications were found (Fig. 1a, b). Our neurological examination revealed bilateral weakness of dorsal and plantar foot flexion; reduced patellar and Achilles’ deep tendon reflexes, with prevalence on the right side; extensor plantar reflex bilaterally; and a hypoesthesia on the left L5–S1 dermatome. Moreover, he had a steppage gait prevalent on the left side. MRI of the dorso-lumbar spine showed multiple subarachnoid cystic lesions constricting the spinal cord and determining parenchymal damage at the level of D6–D8 and D10–D11 and dislocation of cauda roots (Fig. 1c, d). MRI of the lumbo-sacral plexus was unremarkable except for a T2-hyperintense and gadolinium-enhancing lesion of the right gluteus muscle. EMG confirmed a chronic motor multiradiculopathy, with a prevalent involvement of the left L5 and both S1 roots. Sensory and motor-evoked potentials were normal. CSF analysis revealed increased proteins, a moderate number of nucleated cells and a mild decrease in glucose. Serologic and CSF analyses ruled out brucellosis, Lyme disease, syphilis and tuberculosis infections. Dorsal spinal cord was surgically decompressed. Histopathological examination of the biopsied cysts was consistent with neurocysticercosis (NCC) (Fig. 1e, f). The patient was therefore started on treatment with albendazole (15 mg/kg/day) and oral steroids (prednisone 0.5–1 mg/kg/day). In the following months, back pain and gait disturbances recovered, leaving him with a mild weakness of left foot dorsiflexion, whereas no significant modifications were seen on repeat MRI. Two years later, while tapering steroids long lasting attacks of diffuse, oppressive headache without neck stiffness reappeared. Brain MRI revealed normal pressure hydrocephalus secondary to chronic meningitis (Fig. 1g, h). Therapy with oral steroids led to clinical and neuroradiological remission. NCC defines the invasion of the central nervous system (CNS) by Taenia Solium, a parasite endemic in developing countries [1] that is again on rise in western Europe during this new millennium for the increase of immigration and traveling [2]. Despite CNS invasion, clinical manifestations of NCC may remain for a long time silent or fluctuate over time [3]. This accounts for the fact that in developed countries, diagnosis is often delayed, as other more common differential diagnoses are first considered [4]. Indeed, the immunological host response, the variable size, location, number and stage of lesions determine a wide temporal and spatial spectrum of NCC clinical manifestations. Headache, being more frequent in extraparenchymal forms of NCC [5], represents after seizures, the second most frequent manifestation in symptomatic patients, and recognizes raised intracranial pressure, hydrocephalous or D. De Feo (&) B. Colombo D. Dalla Libera V. Martinelli G. Comi Department of Neurology, Institute of Experimental Neurology (INSPE), San Raffaele Hospital (IRCCS), Via Olgettina 60, 20132 Milan, Italy e-mail: defeo.donatella@hsr.it